PMID:12837799
| Citation |
Sudom, A, Walters, R, Pastushok, L, Goldie, D, Prasad, L, Delbaere, LT and Goldie, H (2003) Mechanisms of activation of phosphoenolpyruvate carboxykinase from Escherichia coli by Ca2+ and of desensitization by trypsin. J. Bacteriol. 185:4233-42 |
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| Abstract |
The 1.8-A resolution structure of the ATP-Mg(2+)-Ca(2+)-pyruvate quinary complex of Escherichia coli phosphoenolpyruvate carboxykinase (PCK) is isomorphous to the published complex ATP-Mg(2+)-Mn(2+)-pyruvate-PCK, except for the Ca(2+) and Mn(2+) binding sites. Ca(2+) was formerly implicated as a possible allosteric regulator of PCK, binding at the active site and at a surface activating site (Glu508 and Glu511). This report found that Ca(2+) bound only at the active site, indicating that there is likely no surface allosteric site. (45)Ca(2+) bound to PCK with a K(d) of 85 micro M and n of 0.92. Glu508Gln Glu511Gln mutant PCK had normal activation by Ca(2+). Separate roles of Mg(2+), which binds the nucleotide, and Ca(2+), which bridges the nucleotide and the anionic substrate, are implied, and the catalytic mechanism of PCK is better explained by studies of the Ca(2+)-bound structure. Partial trypsin digestion abolishes Ca(2+) activation (desensitizes PCK). N-terminal sequencing identified sensitive sites, i.e., Arg2 and Arg396. Arg2Ser, Arg396Ser, and Arg2Ser Arg396Ser (double mutant) PCKs altered the kinetics of desensitization. C-terminal residues 397 to 540 were removed by trypsin when wild-type PCK was completely desensitized. Phe409 and Phe413 interact with residues in the Ca(2+) binding site, probably stabilizing the C terminus. Phe409Ala, DeltaPhe409, Phe413Ala, Delta397-521 (deletion of residues 397 to 521), Arg396(TAA) (stop codon), and Asp269Glu (Ca(2+) site) mutations failed to desensitize PCK and, with the exception of Phe409Ala, appeared to have defects in the synthesis or assembly of PCK, suggesting that the structure of the C-terminal domain is important in these processes. |
| Links | |
| Keywords |
Binding Sites; Calcium/metabolism; Crystallography, X-Ray; Enzyme Activation; Escherichia coli/enzymology; Escherichia coli/genetics; Magnesium/metabolism; Manganese/metabolism; Models, Molecular; Mutagenesis, Site-Directed; Phosphoenolpyruvate Carboxykinase (ATP)/chemistry; Phosphoenolpyruvate Carboxykinase (ATP)/genetics; Phosphoenolpyruvate Carboxykinase (ATP)/isolation & purification; Phosphoenolpyruvate Carboxykinase (ATP)/metabolism; Plasmids/genetics; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Pyruvic Acid/metabolism; Trypsin/metabolism |
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